Protein kinases (PKs) are enzymes that play a key role in the regulation of protein functions in living cells. There are 538 protein kinase-encoding genes in human genome [Schwartz et al., Bioorg. Chem. 39 (2011) 192] and it has been estimated that the activity of one third of proteins is regulated by phosphorylation. More than 400 human diseases (including several forms of cancer) have been linked to aberrant protein kinase signaling. This has made PK an important drug target [Cohen, Nat. 20 Rev. Drug Discov. 1 (2002) 309; Fischer, Curr. Med. Chem. 11 (2004) 1563; Schwartz et al., Bioorg. Chem. 39 (2011) 192].
Three kinds of active site-targeted inhibitors of protein kinases are known. The first type of inhibitor is targeted to the nucleotide binding site of PK. Since the nucleotide binding pocket of protein kinases is highly conserved, the development of selective inhibitors of this type is problematic. In addition, these inhibitors have to compete with a high concentration of ATP in the cellular milieu. The second type of active site targeted inhibitors of PKs comprise compounds that associate with the peptide/protein binding site of PK [Bogoyevitch et al., Biochim. Biophys. Acta. 1754 (2005) 79; Lawrence, Handb. Exp. Pharmacol. 167 (2005) 11-44]. The third type is bisubstrate inhibitors that simultaneously associate with the nucleotide and peptide/protein binding sites [recent review: Uri et al., Biochim. et Biophys. Acta 1804 (2010) 541]. Bisubstrate (or biligand) inhibitors have been constructed by combining two fragments, nucleotide analogue or small molecule nucleotide-competitive inhibitor targeted to the nucleotide-binding pocket of PK, and peptide or peptide mimetic, targeted to the peptide/protein binding site of PK. These two fragments are covalently conjugated via a linker, which allows effective association of both of these fragments with the active site of PK. Bisubstrate inhibitor approach could lead to enhanced specificity and potency of inhibition. The most potent bisubstrate inhibitors described are ARC-type inhibitors developed by the authors of the present invention, which show subnanomolar to picomolar potency towards basophilic protein kinases [U.S. Pat. No. 8,158,376; EE200300187; Enkvist et al., J. Med, Chem. 49 (2006) 7150; Viht et al., Anal. Biochem., 362 (2007) 268; Enkvist et al., Bioorg. & Med. Chem. Lett. 19 (2009) 6098; Lavogina et al. J. Med. Chem. 52 (2009) 52, 308; Lavogina et al. Biochim. et Biophys. Acta 1804 (2010) 1857; Enkvist et al. ACS Chem Biol. 10 (2011) 1052]. These inhibitors are constructed by conjugating ATP binding site targeted adenosine-5′-carboxylic acid (Adc) or ATP-competitive inhibitor and the protein substrate domain directed oligo-(L-arginine) or oligo-(D-arginine) via a hydrophobic linker.
Protein kinase CK2 is an acidophilic serine/threonine kinase, which regulates a number of cellular processes. The activity of CK2 is involved in cell growth, proliferation, angiogenesis and suppression of apoptosis, making the kinase a potential target for cancer chemotherapy [Trembley et al., Cell. Mol. Life Sci. 66 (2009) 1858]. In cells, CK2 is mostly present in the form of the holoenzyme, a hetero-tetramer composed of two catalytic (αand/or α′) and two regulatory (β) subunits [Salvi et al., FEBS Lett., 580 (2006) 3948; Niefind et al., EMBO J. 20 (2001) 5320].
Several selective ATP-competitive inhibitors of CK2 have been developed [Cozza et al., Curr. Med. Chem. 20 (2013) 671]. A highly potent and orally available nucleotide-competitive inhibitor CX-4945 is in clinical trials for cancer treatment [Pierre et al., J. Med. Chem. 54 (2011) 635]. Non-ATP-competitive inhibitors of CK2 [Laudet et al., Biochem. J. 408 (2007) 363; Moucadel et al., Oncotarget, 2 (2011) 997] and biligand inhibitors with modest micromolar inhibitory potency [Swider et al., Moll. Cell. Biochem. 356 (2011) 117] have also been described but the inhibitory potency of the disclosed compounds is too low for practical applications.
The evaluation of the structure and functioning of protein kinases and development of potent inhibitors as drug candidates and biomedical research tools requires sensitive detection methods. The majority of kinase inhibitors are evaluated by their inhibitory potencies (IC50) in kinetic studies. The radiometric assay that is based on transfer of radioactively labeled phosphoryl group from [γ-32P] ATP to peptide or protein substrate has been considered the gold standard format because of high sensitivity and direct readout of the catalytic activity of kinase. However, such assay involves labour intensive separation steps and is hazardous due to radioactivity. Fluorometric methods have been developed that are better spatially and temporally focused than radiometric methods, and as such, are better suited for HTS applications. One type of such fluorometric assays is based on the capture of the product of the phosphorylation reaction by an antibody or other macromolecule (e.g., IMAP-particle) that changes the fluorescent properties of the label attached to the substrate of the phosphorylation reaction or that displaces fluorescent reporter molecule from the complex with the macromolecule, changing fluorescence properties such as intensity or anisotropy. Although better suited for HTS format, these types of assay still require effective substrate for the phosphorylation reaction and high-affinity capture particles. An alternative way to characterize the inhibitors of protein kinases is by their binding affinities to the active sites of the enzymes. The dissociation constants (Kd) for the inhibitor-kinase complexes are independent of the Km values of the substrates and are thus better comparable for different kinases using different assay setups. These assay formats utilize a fluorescent reporter molecule—fluorescent probe—that changes its fluorescence properties intensity, anisotropy, lifetime) upon binding to the active site of the PK. Competitive inhibitors displace the probe from the complex with protein kinase resulting in the change of fluorescence characteristics. In addition to the characterization of inhibitors of protein kinases, the fluorescent probes can be applied for detection and quantification of the active forms of PKs in enzyme preparations and biological compositions (cell lysates, cells or tissues).
Although several high-affinity fluorescent probes for protein kinases have been described [Chen et al., J, Biol, Chem. 268 (1993) 15812, WO2005/033330, US2006/0263841], no such examples for protein kinase CK2 have been reported. Generic high-affinity fluorescent probes for protein kinases have been described by the authors of the present invention [U.S. Pat. No. 8,158,376; Vaasa et al. Anal. Biochem. 385 (2009) 85-93; Enkvist et al. ACS Chem. Biol. 10 (2011) 1052], but these compounds have shown selectivity towards basophilic protein kinases and no binding to protein kinase CK2 with these probes have been observed.